Saponin containing extracts prepared from hesperaloe useful in the treatment of non-human animals

ABSTRACT

Disclosed are animal feed compositions comprising a saponin extracted from a non-woody plant of the genus  Hesperaloe . The saponin containing extracts, which generally constitute the water soluble fraction, are useful as additives to basal animal feed to reduce the production of ammonia. In certain embodiments, saponin containing compositions of the present invention may inhibit proteolytic microorganisms, reducing ammonia and improving animal welfare and animal performance. In other embodiments, administration of saponin containing compositions of the present invention to non-human animal inhibits the release of gaseous ammonia through the inhibition urease. The compositions of the present invention may be administered to non-human animals, particularly poultry, to reduce ammonia emissions. In certain embodiment, the animal feed composition comprises a basal animal feed and an extract from a non-woody plant of the genus  Hesperaloe , the extract comprising 25(27)-dehydrofucreastatin, 5(6), 25(27)-disdehydroyuccaloiside C, 5(6)-disdehydroyuccaloiside C, furcreastatin, yuccaloiside, or a mixture thereof.

BACKGROUND

Plants produce a vast and diverse assortment of organic compounds, thegreat majority of which do not appear to participate directly in theirgrowth and development. These substances, traditionally referred to assecondary metabolites or plant natural products, often are distributedamong limited taxonomic groups within the plant kingdom. The functionsof secondary metabolites remain largely unknown, although a number ofcompounds have been associated with attributes useful to the plants e.g.protection against herbivores and protection against microbialinfection, as attractants for pollinators and seed-dispersing animals,and as compounds that influence competition among plant species(allelochemicals). There is a growing interest in plant naturalproducts, since these products often have a wide range of applicationsin different kinds of industries, including pharmaceutical industries,cosmetic industries, food industries, detergent industries, and thelike.

A particular group of plant secondary metabolites of interest aresaponins. Saponins are glycosylated compounds classified as eithertriterpenoids, steroids, or steroidal glycoalkaloids. Saponins consistof one or two sugar moieties which are coupled to the aglycon (mono- andbisdesmosides, respectively). Saponins can be hydrolyzed to sapogeninsand sugar moieties by acid hydrolysis or enzymatic methods. Saponins aregenerally water soluble high molecular weight compounds with molecularweights ranging from 600 to more than 2,000 daltons.

The asymmetric distribution of their hydrophobic (aglycone) andhydrophilic (sugar) moieties confers an amphipathic character to thesecompounds which are largely responsible for their detergent-likeproperties. The ability of lowering surface tension make saponinspotentially well suited for use in the cosmetic and in the detergentindustries.

Saponins also have the ability of forming insoluble complexes withcholesterol, which makes some of them suitable for use in thepharmaceutical industry as cholesterol lowering agents. Other saponinsare associated with formation of immunostimulating complexes that areuseful in vaccine strategies.

Currently, a major limitation to the broad exploitation of saponins isthe fact that commercially available saponins are relatively expensive.The expenses is due in large part to the limited number of plantextracts having significant amounts of saponins. Currently, commerciallyavailable plant extracts containing saponins include Saponariaofficinalis, Quillaia bark and stem, Castanea sativa seeds, and extractsof various Yucca species.

Plant extracts containing saponins are thus of general interest within awide range of different industries. There is therefore a growing need inthe art for alternative sources of saponin extracts and these plantsources should preferably be cheap, easy to obtain, and preferably thesaponin content should be relatively high.

SUMMARY

The present inventors have now discovered that water soluble solidsextracted from non-woody plants of the genus Hesperaloe comprise one ormore saponins that may be useful as additives to basal animal feed toreduce the production of ammonia. In certain embodiments, saponincontaining compositions of the present invention may inhibit proteolyticmicroorganisms, reducing ammonia and improving animal welfare and animalperformance. In other embodiments, administration of saponin containingcompositions of the present invention to non-human animal inhibits therelease of gaseous ammonia through the inhibition urease, which catalyzehydrolysis of urea into carbon dioxide and ammonia resulting in therelease of gaseous ammonia.

Accordingly, in one embodiment, the present invention provides novelpharmaceutical, dietary supplements and food ingredient compositionsthat are safe for non-human animal consumption, reduce ammoniaemissions, have beneficial health effects, and can enhance the growthanimals treated therewith.

In other embodiments, the present invention provides novelpharmaceutical, dietary supplements and food ingredient compositionscomprising at least one component selected from the extract(s),fraction(s), active compound(s) and phytochemical(s) or mixtures thereofderived from non-woody plants of the genus Hesperaloe including, forexample, Hesperaloe funifera, Hesperaloe nocturne, Hesperaloeparviflora, and Hesperaloe chiangii. The novel pharmaceutical, dietarysupplements and food ingredient compositions may optionally contain oneor more of pharmaceutically and dietetically acceptable phytochemicalactives, diluents, vehicles, carriers and actives or mixtures thereof.

In a particularly preferred embodiment, the present invention providesan animal feed additive comprising water soluble solids derived fromnon-woody plants of the genus Hesperaloe. Preferably the water solublesolids comprise at least about 2 wt % saponin, more preferably at leastabout 5 wt % saponin and still more preferably at least about 10 wt %saponin, such as from about 2 to about 30 wt % saponin, such as fromabout 5 to about 25 wt % saponin, wherein the weight percentages arebased upon the total bone dry weight of the water soluble solids. In aparticularly preferred embodiment, the present invention provides ananimal feed composition comprising a basal animal feed and a mixture ofsaponins derived from non-woody plants of the genus Hesperaloe. Incertain embodiments the mixture of saponins may comprise one or more of25(27)-dehydrofucreastatin (FIG. 2A), 5(6),25(27)-disdehydroyuccaloisideC(FIG. 2B), 5(6)-disdehydroyuccaloiside (FIG. 2C), furcreastatin oryuccaloiside C.

In other embodiments the present invention provides methods of treating,controlling or preventing of one or more conditions in non-humananimals, especially birds and more particularly poultry by administeringthereto a pharmaceutical, dietary supplement or food ingredientcomposition comprising at least one component selected from theextract(s), fraction(s), active compound(s) and phytochemical(s), ormixtures thereof, derived from non-woody plants of the genus Hesperaloe.For example, saponin containing compositions derived from Hesperaloebiomass may be administered to non-human animals to reduce environmentalammonia and odor. In a particularly preferred embodiment, the saponincompositions of the present invention may be administered orally topoultry to reduce environmental ammonia and odor.

In another embodiment the present invention provides an animal feedcomposition comprising basal animal feed; and a water solublecomposition extracted from a non-woody plant of the genus Hesperaloe andcomprising at least one saponin. In a particularly preferred embodimenttotal amount of saponin, based upon the total mass of feed, ranges fromabout 1.0 to about 30.0 grams per 100 kilograms of feed.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate a triterpenoid saponin and a steroidalsaponin, respectively; and

FIGS. 2A-C illustrate various novel saponins extracted from non-woodyplants of the genus Hesperaloe according to the present inventionincluding 25(27)-dehydrofucreastatin (FIG. 2A),5(6),25(27)-disdehydroyuccaloiside C (FIG. 2B), and5(6)-disdehydroyuccaloiside C (FIG. 2C).

DEFINITIONS

As used herein the term “biomass” generally refers to whole plants andplant organs (i.e., leaves, stems, flowers, roots, etc.) of the genusHesperaloe including, for example, Hesperaloe funifera, Hesperaloenocturne, Hesperaloe parviflora, and Hesperaloe chiangii.

As used herein the term “bagasse” generally refers to biomass that hasbeen cut to size and then subjected to high pressure so that theresulting material has less liquid than the biomass from which it isderived. High pressure can be achieved by using compression pressure,such as that provided by machines such one or more opposedcounter-rotating rolls, a mechanical press, a screw press as well as bydirect hydraulic pressure and other processes to apply pressure to thebiomass and remove intercellular and intracellular liquid.

As used herein the term “milling” generally refers to the application ofsufficient pressure to force the intercellular and intracellular liquidfrom the biomass.

As used herein the term “saponin” generally refers to glycosidescomprising a sugar component referred to as a glycone and a non-sugarcomponent referred to as an aglycone. Depending on the structure of theaglycone the saponin may be classified as a triterpenoid saponin,illustrated in FIG. 1A, or to steroidal saponin, illustrated in FIG. 1B.The aglycone portion of the saponin may be either a pentacyclictriterpenoid or a tetracyclic triterpenoid, both of which contain 30carbon atoms. Whether steroidal or triterpenoid, saponins may be mono,bi- or tridesmodic. Monodesmodic saponins have a single saccharide,normally attached at C-3. Bidesmodic saponins have two saccharides,often with one attached through an ether linkage at C-3 and the othereither attached through an ester linkage at C-28 or through an etherlinkage at C-20 (pentacyclic and tetracyclic triterpene saponins,respectively), or through an ether linkage at C-26 (furostane saponins).

As used herein the term “water soluble solids” generally refers to drymatter which remains after the extract has been centrifuged, filteredand all water is evaporated. The procedure for measuring water solublesolids of a biomass extract of the present invention is described indetail in the Test Methods section below. Water soluble solids may beexpressed on a percentage basis relative to the mass of bone drybiomass.

As used herein the term “water insoluble solids” generally refer to thefraction of extract that is removed by centrifugation and filtration inthe course of measuring water soluble solids, as described in the TestMethods section below.

As used herein, “reducing ammonia emissions” from a non-human animaltreated with saponin containing composition of the present inventionrefers to reducing emitted ammonia gas relative to an untreatednon-human animal. In some embodiments, the reduction is from about 10 toabout 30% in ammonia emissions when compared to an untreated animal. Insome embodiments, the reduction is from about 15 to about 25% in ammoniaemissions when emissions are standardized to animal live weight. In someembodiments, the reduction of ammonia emissions from the animal does notsignificantly negatively affect the animal, such as, for example,lowering body weight, or decreasing eating quality.

As used herein, “reducing carbon dioxide emissions” from a non-humananimal treated with saponin containing composition of the presentinvention refers to reducing emitted carbon dioxide gas relative to anuntreated animal. In some embodiments, the reduction is about 5% incarbon dioxide emissions when compared to an untreated animal whencarbon dioxide emissions are standardized to animal live weight.

The term “effective amount”, in the context of administration, refers tothe quantity of saponin administered to a non-human animal in need oftreated, which amount is sufficient to reduce ammonia or carbon dioxideemissions from the animal as compared to an untreated animal.

DETAILED DESCRIPTION

The present invention relates to novel pharmaceutical, dietarysupplements and food ingredient compositions comprising at least onecomponent selected from the extract(s), fraction(s), active compound(s)and phytochemical(s) or mixtures thereof derived from non-woody plantsof the genus Hesperaloe including, for example, Hesperaloe funifera,Hesperaloe nocturne, Hesperaloe parviflora, and Hesperaloe chiangii,optionally containing one or more of pharmaceutically and dieteticallyacceptable phytochemical actives, diluents, vehicles, carriers andactives or mixtures thereof. In a particularly preferred embodiment, thepresent invention provides an animal feed additive comprising watersoluble solids derived from non-woody plants of the genus Hesperaloe.

The novel saponin containing pharmaceutical, dietary supplements andfood ingredient compositions may be administered to a non-human animalto reduce gas emissions, particularly ammonia and carbon dioxide. Thecompositions of the present invention are particularly well suited forthe treatment of non-human-animals including, for example, bovine, fowl,porcine, ovine, and equine species. By way of example, the methods andcompositions of the invention can be used for the treatment of cattle,chickens, turkeys, ducks, quail, geese, pigs, and sheep. In aparticularly preferred embodiment, the methods and compositions of thepresent invention can be used for the treatment of poultry and moreparticularly may be administered to poultry to reduce gas emissions.

In certain embodiments, the pharmaceutical, dietary supplements and foodingredient compositions comprise a mixture of saponins. In this mannerthe compositions of the present invention may comprise least 2 wt %,based upon the bone dry weight of the composition, saponins as measuredby the total saponin assay set forth in the Test Methods section below.In a particular embodiment, the saponin containing composition used inaccordance with the invention comprises at least about 2 wt % saponin,more preferably at least about 5 wt % saponin and still more preferablyat least about 10 wt % saponin, such as from about 2 to about 30 wt %saponin, such as from about 10 to about 25 wt %. It is believed that theeffects of the composition are related to the total amount of saponinspresent. Thus, one of skill in the art will appreciate that if a certainamount of saponins is desired it can be achieved either through varyingthe volume of a certain concentration composition administered, varyingthe concentration of a certain volume of a composition, or both.

Saponins useful in formulating the pharmaceutical, dietary supplementsand food ingredient compositions of the present invention are preferablyextracted from non-woody plants of the genus Hesperaloe. Saponinsderived from Hesperaloe may have at least one of the following aglyconesor genins: kammogenin, manogenin, gentrogenin, hecogenin, tigogenin,sarsapogenin, chlorogenin and gitogenin or their corresponding isomer oroxidized or reduced forms with at least one of the following glycosidicmoieties (in the form of acid or salt): glucose, xylose, rhamnose,arabinose, or galactose. In other embodiments the steroidal saponins maycomprise agamenoside, agaveside, agavoside, magueyside, agavasaponi,cantalasaponin, sisalsaponin, gabrittonoside, dongnoside or amolonin, orother steroidal saponins.

Extractives may be recovered from non-woody plants of the genusHesperaloe by extracting biomass, particularly the leaves and moreparticularly the leaves above the crown of the plant, with at least onesolvent selected from the group consisting of water, methanol, ethanol,butanol, and isopropanol and mixtures thereof. For example, in oneembodiment, the process comprises contacting biomass with an extractantsolution comprising water and separating the water soluble fraction fromthe insoluble biomass fraction. In other embodiments the extractantsolution may comprise, in addition to water, a surfactant, a solvent andoptionally extract-bearing juice. The extract-bearing juice can comefrom, for example, an earlier extraction step or an earlier millingstep.

A simple water extraction of Hesperaloe biomass may yield a crudeaqueous extract comprising saccharides, polysaccharides, inorganicsalts, saponins and sapogenins. A crude extract may also be producedusing methanol as a solvent, or a mixture of methanol and water, toextract biomass, which may have been previously extracted with acetoneor diethyl ether to remove lipids and pigments. In other instances, thebiomass may be extracted with a 4:1 ethanol-water solvent, followed bysubsequent defatting of the extract with a non-polar solvent such ashexane. In certain instances, the defatted extract may be subjected tofurther treatment to isolate specific water soluble components, such assaponins, which may be purified from the defatted extract by mixing withbutanol and separating the butanol phase to yield a mixture of saponinsthat are substantially free from proteins and free saccharides andpolysaccharides.

Hot aqueous extractants can also be used. For example, in one embodimentwater soluble solids may be extracted from Hesperaloe biomass,particularly the leaves, by extracting the biomass with hot aqueousethanol or isopropanol (75 to 95% by weight alcohol). The aqueousalcohol extraction fluid may then be filtered and concentrated, and thefat-soluble material may be removed by mixing the extraction fluid witha non-polar solvent such as hexane. A substantially pure saponincomposition may then be prepared by further extracting defatted extractwith a polar solvent such as butanol.

For the purpose of preparing the compositions of the present invention,and for use in the present method, a simple aqueous extract may bepreferred, although other extraction methods are within the scope of thepresent invention. In a particularly preferred embodiment, Hesperaloebiomass may be cut to size, pressed, and extracted with an aqueoussolvent to remove water soluble extracts such as inorganic salts,saccharides, polysaccharides, organic acids and saponins. The watersoluble extracts are collected and may be concentrated by techniqueswell known in the art such as, for example, evaporation, spray-drying,drum drying and the like. The extract may be concentrated until it has asolids content of about 20 to about 100% solids by weight, such as fromabout 20 to about 95% solids by weight, such as from about 20 to about80% solids by weight.

In a particularly preferred embodiment water soluble extracts areconcentrated by spray drying by feeding the extract solution toatomizing equipment. Suitable atomizing equipment includes, but is notlimited to, a rotary wheel atomizer, a pressure nozzle atomizer, and adual fluid nozzle atomizer. Rotary wheel, pressure nozzle and dual fluidnozzle atomizers are known to those of ordinary skill in the art andinclude those in spray dryers commercially available from a variety ofsources, such as GEA Process Engineering.

In certain embodiments biomass may be milled to separate the bagasse andwater soluble solids using a roll, screw, and other forms of presses. Incertain preferred embodiments biomass is passed between one or more nipsof opposed counter-rotating rolls to maximize the mechanical removal ofjuice. The bagasse can then be contacted with the juice in a subsequentmilling step, as will be described more fully below. In certaininstances, the biomass may be cut to size and cleaned prior to milling.Cutting and cleaning may be carried out using well known methods in theart. In a particularly preferred embodiment, the biomass is cleaned toremove debris such as dirt without the use of water or other solvents.

In other embodiments the water soluble solids may be recovered frombiomass by diffusion. In diffusion, the biomass brought into contactwith the liquid to extract the liquid components. Usually, the biomassis prepared by first cutting, but not shearing or crushing so as tominimize the damage to fibers and avoid the creation of an excessiveamount of fines. The prepared biomass is then washed repeatedly, usuallyusing a solvent, to extract the liquid contained in the biomass. Thesolvent can be any of the foregoing solvents. An exemplary treatmentsolvent is water, particularly hot water such as water heated to atemperature from about 40 to about 90° C. The solvent can be circulatedand reused so that the solvent used for a first extraction is reused asa solvent to extract subsequent prepared biomass.

Various types of diffusers are known in the art and can be adapted foruse with biomass as described herein. Suitable diffusers include a ringdiffuser, a tower diffuser, or a drum diffuser. Exemplary diffusionsystems are discussed, for example, in U.S. Pat. Nos. 4,182,632,4,751,060, 5,885,539 and 6,193,805 the contents of which are herebyincorporated in a manner consistent with the present disclosure.Numerous other diffusion methods and devices for the diffusion methodare known and can be adapted for use in the methods described herein.One such diffuser is the continuous-loop, counter-current, shallow-bedCrown Model III Percolation Extractor, commercially available from CrownIron Works, Blaine, Minn.

The biomass, cut or uncut, may be extracted by any suitable extractionprocess as discussed above. In a particularly preferred embodiment, thesolvent used for extraction comprises water. One of skill in the artwill recognize the ratio of extraction solvent to biomass will varybased on the solvent, the amount of biomass to be extracted and theextraction procedure. In certain preferred embodiments, the extractionsolvent is water and the ratio of extraction solvent to biomass, on thebasis of liters of extraction solvent to kilogram of bone-dry biomass,is from about 1:5 to about 1:100, such as from about 1:5 to about 1:50and more preferably from about 1:5 to about 1:20.

The pH of the extraction solvent can be between about pH 5.0 and 8.0,such as, for example, between about pH 6.0 and about pH 8.0, betweenabout pH 6.5 and about pH 7.5. In a particular embodiment, theextraction solvent is water having a pH between about pH 6.5 and aboutpH 7.5. In those embodiments where extraction includes imbibition with acrude juice, the imbibition fluid may have a pH from about 4.0 to about5.0.

The extraction may be carried out at temperatures between about 25 andabout 90° C., such as, for example, between about 30 and about 80° C.,between about 35 and about 75° C., between about 40 and about 70° C.,between about 45 and about 65° C. or between about 50 and about 60° C.

In embodiments where the extraction process is a batch extractionprocess, the duration of extraction may range from about 0.25 to about24 hours, such as, for example, from about 0.5 to about 2 hours, fromabout 1 to about 8 hours, or from about 1 to about 6 hours.

In embodiments where the extraction process is a continuous process, theduration of extraction may range from about 0.25 to about 5 hours, suchas, for example, from about 0.5 to about 3 hours.

After extraction the water insoluble biomass material may be separatedfrom the water soluble solids by filtration to provide a filtratecontaining inorganic salts, saccharides, polysaccharides, organic acidsand saponins (referred to herein as the “first filtrate”). Separationcan be achieved by any suitable means including, but not limited to,gravity filtration, a plate-and-frame filter press, cross flow filters,screen filters, Nutsche filters, belt filters, ceramic filters, membranefilters, microfilters, nanofilters, ultrafilters or centrifugation.Optionally various filtration aids such as diatomaceous earth,bentonite, zeolite, and the like, may also be used in this process.

After separation, the pH of the first filtrate may be adjusted to removeadditional impurities. In one embodiment, the pH of the first filtratecan be adjusted to between about 8.5 and about 10.0 by treatment with abase, such as, for example, calcium oxide or hydroxide (about 1.0% fromthe volume of filtrate) with slow agitation.

In other instances the first filtrate may be concentrated or subjectedto further processing to isolate a particular mixture of saponins orindividual saponins. In other instances, the clarified juice may befurther purified to remove saccharides, polysaccharides, and organicacids to yield composition comprising saponins and being substantiallyfree from remove saccharides, polysaccharides, and organic acids. Theforegoing impurities may be removed by mixing the juice or firstfiltrate with a water-immiscible polar solvent such as alcohols havingfrom 4 to 6 carbon atoms, such as butyl, amyl, hexyl and cyclohexylalcohols. The solvent phase containing the saponin may be subjected tofurther treatment to separate the saponin from the alcohol phase. Thiscan be accomplished in various ways including, for example, by cooling,by dehydrating the solvent extract, or by adding an organic solventwhich is miscible with the alcohol solvent but in which the saponin isinsoluble. Suitable precipitating solvents include, for example, diethylether, petroleum ether, acetone, and chloroform.

In a particularly preferred embodiments, the saponin is separated fromthe alcohol by flash evaporation. Flash evaporation is a technique knownin preparative chemistry for the rapid removal of a volatile componentfrom a liquid mixture. The volatile liquid is removed from solution byrapid conversion to a vapor phase by creating a thin film of thesolution over a large surface area under reduced pressure oftenaccompanied by an increase of temperature of the solution above ambientbut less than the boiling point of the solution at atmospheric pressure.The actual thickness of the film and the area over which it is appliedis chosen to provide optimum evaporation and ease of use, butevaporation may be substantially instantaneous (hence the name “flash”evaporation). Flash evaporation avoids the prolonged use of hightemperatures that may degrade the intended product and has the abilityto remove almost all of the alcohol component (which makes the remainingsolution suitable for the preferred practice of spray drying employed inthe next step. The alcohol may be recovered from this step and reused inthe extraction process.

The saponin content of the alcohol extract can be further increased bypassage over an ultrafiltration membrane without significant alterationto or loss of the saponin composition. This concentrated saponinfraction where the saponin content is in the range of 85-90%, can thenbe further purified in a liquid state or reduced to a dry state.Individual saponins may be recovered by a combination of reversed-phasesolid phase extraction and preparative reversed-phase HPLC.Alternatively, the alcohol extract containing saponins can befractionated directly by a combination of reversed-phase solid phaseextraction and preparative reversed-phase HPLC.

In still other embodiments saponins may be purified from juice preparedaccording to the present invention comprises the steps of mixing thejuice with a salt and a solvent to form a first solution. The solventmay comprise one or more solvents selected from acetic acid, acetone,acetonitrile, benzene, 1-butanol, 2-butanol, 2-butanone, t-butylalcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane,1,2-dichloroethane, diethylene glycol, diethyl ether, diglyme,1,2-dimethoxyethane, dimethylformamide, dimethylsulfoxide, 1,4-dioxane,ethanol, ethyl acetate, ethylene glycol, glycerin, heptane,hexamethylphosphoramide, hexamethylphosphorous triamide, hexane,methanol, methyl-t-butyl ether, methylene chloride,N-methyl-2-pyrrolidinone, pentane, perchloroethylene, petroleum ether,1-propanol, 2-propanol, pyridine, tetrahydrofuran, toluene,triethylamine, trifluorotoluene, water, xylene, or any combination ofthe forgoing. In some embodiments the solvent is water. The salt may beselected from an alkali metal salt, an alkaline earth salt, a transitionmetal salt, an ammonium salt, or combinations of the forgoing. Incertain preferred embodiment the salt added to the plant extract to formthe solution is an alkaline earth metal salt. In particularly preferredembodiments the salt is calcium chloride (CaCl₂)), magnesium chloride(MgCl₂), or a mixture thereof.

The pH of the first solution is generally adjusted to a pH from about6.0 to about 9.0, such as from about 6.0 to about 8.0, such as fromabout 6.0 to about 7.0. At least one phosphate may then be added to thefirst solution to form an ion-polysaccharides complex precipitate.Useful phosphates include, for example, sodium hydrogen phosphate(Na₂HPO₄), sodium dihydrogen phosphate (NaH₂PO₄), sodium phosphate(Na₃PO₄), or sodium hydrogen bisphosphate (Na₂H₂PO₇).

The precipitated ion-polysaccharides complex may be removed byfiltration to yield a second solution, which may be further clarified toproduce an extract of purified saponins. Optionally, the extract can beconcentrated by any filtration technique known in the prior art.Preferably, the concentration of the extract of purified saponins iscarried out by nanofiltration, ultrafiltration and diafiltration, or anycombination of these techniques. In some embodiments, the saponinextract is substantially free of proteins. In some embodiments, thesaponin extract is substantially free of polysaccharides. In someembodiments, the saponin extract is substantially free of phenoliccompounds.

The amount of water soluble solids recovered from biomass may varydepending on the extraction efficiency, however, in certain instancesfrom about 100 to about 400 grams of water soluble solids may beextracted per kilogram of bone dry biomass, such as from about 120 toabout 350 grams per kilogram, such as from about 150 to about 300 gramsper kilogram. Of the extracted water soluble solids, the total saponinsmay comprise at least about 5 wt %, such as at least 10 wt %, such as atleast 20 wt %, such as from about 5 to about 40 wt %, such as from about10 to about 30 wt %, based upon the bone dry weight of the water solublesolids. In certain instances the amount of total saponins that may beextracted from biomass may range from about 10 to about 400 grams perbone dry kilogram of biomass, such as from about 20 to about 300 grams,such as from about 25 to about 200 grams, such as from about 10 to about100 grams. In certain instances, the amounts of materials (on bone drygrams per kilogram of bone dry biomass) removed from the biomass duringthe extraction process may range as set forth in Table 1, below.

TABLE 1 Amount (g/kg of bone dry biomass) Total Extracted Solids100-400  Total Water Insoluble Solids 5-50 Total Water Soluble Solids95-350 Total Saponins  5-160

The total amount of saponins that may be extracted from Hesperaloebiomass according to the present invention may range from about 10 toabout 100 grams per bone dry kilogram of biomass, such as from about 20to about 80 grams, such as from about 25 to about 75 grams. The saponinsmay be provided as part of a crude juice, as part of a dried watersoluble solids compositions, as a partially purified compositions or asa substantially pure composition comprising a mixture of saponins.

In certain embodiments saponins extracted from Hesperaloe biomass maycomprise one or more saponins selected from 25(27)-dehydrofucreastatin(FIG. 2A), 5(6),25(27)-disdehydroyuccaloiside (FIG. 2B),5(6)-disdehydroyuccaloiside (FIG. 2C), furcreastatin and yuccaloiside.

Compositions useful in the present invention may be prepared by blendingan aqueous extract from Hesperaloe biomass with one or more polyhydroxyalcohols including glycerol, propylene glycol, polyalkylene glycol suchas polyethylene glycol and polypropylene glycol, and polyglycerol.Preferred polyhydroxy alcohols have less than about eight carbon atoms.Glycerol and propylene glycol are particularly preferred polyhydroxyalcohols.

The composition may also comprise saccharides, which may be present inthe aqueous extract or may be added after extraction during formulation.Saccharides useful in compositions of the present invention includemonosaccharides such as glucose, disaccharides such as sucrose andpolysaccharides such as starch.

In still other embodiments compositions in accordance with embodimentsof the invention can include various other additives known in the art tohave benefits for the maintenance and well-being of non-human animals.By way of example, compositions can also include components such asVitamin E, Vitamin A Propionate, Vitamin A Palmitate, Vitamin B1,Vitamin B2, Vitamin B6, Vitamin B12, D-Activated Animal Sterol (sourceof Vitamin D3), yeast components, dried egg solids, dried casein, anddried whey.

Saponin containing compositions of the present invention may be inliquid or dry forms. By way of example, a saponin containing Hesperaloeextract may be dried into a powder form. In this form, the saponincontaining composition may be administered to an animal as a pill orbolus or mixed in with other components such as a feed ration. Forexample, dry powder formulations of saponin containing compositions maybe added to the feed ration via a micro-ingredient machine or added to afeed mix truck and mixed thoroughly to assure even distribution in thefeed. Saponin containing Hesperaloe extract may also be in liquid formwith an amount of a carrier liquid such as water. In this form, thesaponin containing composition may be administered to an animal as aliquid drench.

Saponin containing compositions of the present invention may beadministered to non-human animals in need there of as a single dose, asmultiple doses as part of a feeding regiment. For example, a non-humananimal may receive an initial dose and then receive subsequentmaintenance doses in lesser amounts. A non-human animal may receivemultiple doses of a saponin containing composition in one day or mayreceive multiple doses over multiple days.

In certain embodiments, non-human animals, especially birds and moreparticularly poultry, can be treated with a saponin containingcomposition in an amount that is effective to improve the feedingcharacteristics of an animal and/or improve the productioncharacteristics of an animal in comparison to an untreated controlanimal. Production characteristics can include carcass quality grades,yield grades, average daily gain, and the like. In an embodiment, whenadministered as a feed additive to poultry, the amount of saponins per100 kilograms of feed is preferably at least about 1 g/100 kg, such asfrom about 1 to about 50 g/100 kg, such as from about 2 to about 40g/100 kg.

In particularly preferred embodiment the administration of saponins to anon-human animal in need thereof is carried out by including thesaponins in an animal feed. The basal animal feed may be a dry feed, ora liquid feed and the compositions of the present invention may beformulated as a liquid, a slurry, a dry powder, a dry granular mix, apaste, a pellet, or a block. Suitable feeds may be prepared by applyingsaponins containing extracts, such as my mixing or spraying, withsuitable basal animal feeds commonly employed in the feeding of animals.Typical basal animal feeds useful in the present invention may compriseone or more of corn meal, corncob grits, soybean meal, alfalfa meal,rice hulls, soybean mill run, cottonseed oil meal, bone meal, groundcorn, corncob meal, wheat middlings, limestone, dicalcium phosphate,sodium chloride, urea, distillers dried grain, vitamin and/or mineralmixes, cane molasses or other liquid carriers and the like. Such basalanimal feeds promote a uniform distribution and administration of thesaponins.

In still other embodiments saponin containing extracts of Hesperaloe maybe formulated as urease inhibiting compositions. The as ureaseinhibiting compositions of the present invention may comprise an amountof Hesperaloe extract sufficient to provide urease inhibiting activity.In certain instances, when administered to inhibit urease, the totalsaponins in the composition is at least about 1 wt %, and morepreferably at least about 2 wt %, and still more preferably at leastabout 5 wt %, based on the total weight of the composition. In certainpreferred embodiments the urease inhibiting compositions of the presentinvention comprise one or more saponins extracted from Hesperaloe wherethe saponins include 25(27)-dehydrofucreastatin (FIG. 2A),5(6),25(27)-disdehydroyuccaloiside (FIG. 2B),5(6)-disdehydroyuccaloiside (FIG. 2C), furcreastatin or yuccaloiside.

Urease inhibiting compositions may be provided as animal feed, therebysimplifying dosing. The compositions may include a saponin compositionextracted from Hesperaloe and an animal feedstuff, such as thosediscussed above, and may be formulated in various ways. For exampleurease inhibiting compositions can be formulated as a liquid, slurry,dry powder, dry granular mix, paste, pellets, block, or the like.Compositions may be administered to an animal as a pill, a bolus, or aliquid drench. In certain instances urease may be inhibited, and ammoniaemissions reduced, by administering an effective amount of saponinderived from Hesperaloe along with the animal's feed ration. Forexample, in some embodiments, a composition containing saponin derivedfrom Hesperaloe, such as a liquid composition, can be mixed in with ananimal's water.

Test Methods Water Soluble Solids

Total biomass water soluble solids may be determined using anAccelerated Solvent Extraction system (ASE) such as a Dionex™ ASE™ 350(Thermo Fisher Scientific, Waltham, Mass.). Approximately 10 grams ofharvested biomass is dried to a constant weight in an oven, typically 4hours at 125° C. After drying 1.5-2.0 grams of the bone dry biomass isaccurately weighed and the weight (W_(b)) recorded to the nearest 0.001gram. Using water as the solvent, biomass is extracted using theconditions set forth in the table below. The ratio of biomass to solventis generally 21:1 and five consecutive water extraction cycles areperformed. At the end of each extraction cycle, the liquid phase iscollected, dried under vacuum at approximately 40° C. and the weight ofthe dried material (W_(i)) is recorded to the nearest 0.001 g. The totalweight of water soluble solids (W_(e)) is calculated by summing theweight of solids recovered from each extraction cycle (W_(i)). Totalwater soluble solids as a percentage of bone dry biomass is thendetermined using the following equation: Water Soluble Solids (wt%)=W_(e)/W_(b)*100.

Pressure (psi) 1500 Temperature (° C.) 40 Static Time (min.) 10 Cycles(no.) 5

The total water soluble solids in biomass extract may be determined bywithdrawing an appropriate aliquot, typically about 10-50 ml,transferring to clean, pre-weighed centrifuge tube. The tube is weighedto the nearest 0.001 gram and centrifuged at 7000 rpm for 20 minutes.The weight of extract (W₁) is calculated. The supernatant was thentransferred to clean, pre-weighed beaker (D₀), and weighed. The beakercontaining the sample is then placed at 140° C. in a hot air oven forovernight drying. The beaker is removed from the oven and desiccated tocool to room temperature then weighed to the nearest 0.001 gram (D₁).The weight percentage of soluble solids, based upon the weight of theextract, is determined using the formula below:

${{Water}{Soluble}{Solids}\left( {{wt}\%} \right)} = \frac{\left( {D_{1} - D_{0}} \right) \times 100}{W_{1}}$

D₁=mass of empty beaker+dried soluble solids, D₀=mass of empty beaker,W₁=initial bone dry mass of biomass.

Total Saponins

Total saponins were measured generally as described in Makkar, HarinderP. S., Sidhuraju, P., Becker, Klaus (2007) Plant Secondary Metabolites,chapter 17, pp 93-100. A standard saponin solution was prepared byweighing 10 mg of diosgenin (MilliporeSigma >93%), dissolving in 16 mLof methanol and adding 4 mL of distilled water. The solution was mixedthoroughly to yield a 0.5 mg/mL diosgenin solution in 80% methanolsolvent. The standard was used to produce a calibration curve bytransferring various amounts of the standard (0, 10, 20, 40, 60, 80, and100 μL) into 13-mm glass test tubes. A solution of 80% aqueous methanolwas added to a total volume of 100 μL.

Prior to testing samples of biomass extract were adjusted to about 0.5wt % total solids by dilution with water to ensure absorbency resultfell along the saponin standard calibration curve range. Samples ofdiluted extract (20-μL) were pipetted into 13-mm glass test tubes andthe volume was brought up to 100 μL with 80 μL methanol. Each sample wastested in triplicate.

To each sample 100 μL of vanillin reagent (prepared by dissolving 800 mgof vanillin in 10 mL of 99.5% ethanol (analytical grade)) and then 1.0mL of 72% (v/v) sulfuric acid (72% (v/v) sulfuric acid prepared byadding 72 mL of sulfuric acid (analytical grade, 95%, w/w) to 28 mL ofdistilled water) were added. Solutions were mixed well and heated at 60°C. for 10 minutes. Samples were then cooled in an ice bath and 1 mL ofsolution was transferred into respective cuvette and absorbance at 544nm was read. The total mass of saponins in the sample may be calculatedbased upon the standard absorbency curve as follows:

Saponin (μg)=[Slope]×Measured Absorbency−[Intercept]

Examples

The inventive extract was prepared by forage harvesting matureHesperaloe funifera leaves above the crown, cutting the leaves intopieces ranging from about 0.50 to about 8.0 cm and pressing the cutbiomass using a sugar cane tandem press, each mill of the tandem presshaving 3-rollers. The biomass was passed through the tandem mill threetimes. Imbibition water was added prior to first mill in the tandem. Thecrude juice was collected and passed through 25 mm filter and heated toboiling in a flat pan evaporator (Leader Evaporator Company, Swanton,Vt.) to concentrate the extract to 29% solids. The water-soluble solidscomprised 21 wt % total saponins, based upon the bone dry weight ofwater soluble solids. The concentrated juice was diluted with water toprovide a composition comprising 5 wt % total saponins.

For comparison a composition containing 5 wt % total saponins extractedfrom Yucca was prepared. The Yucca derived saponin are marketed underthe tradename FOAMATION™ (commercially available from Ingredion,Westchester, Ill.) and were provided as 50 wt % total saponins.FOAMATION™ was diluted with water to provide a composition comprising 5wt % saponins.

To test the efficacy of urease inhibitors, 2.0 mL of a putativeinhibitor and 2.0 mL Jack Bean urease (diluted to 10 mg/mL) (SigmaDiagnostics, St. Louis, Mo., Sigma U-4002) were added to a cuvette. Tothis mixture, 18 mL of urine is added to the cuvette and the mixture isincubated for 2 hours. The liberation of ammonia was determined usingGastec's standard ammonia (NH3) detector tube system (Model 1M 003MJ1,supplied by Gastec Corporation, Kanagawa, Japan). The reduction inammonia achieved with the inventive compositions is shown in Table 2,below.

TABLE 2 Codes Inhibitor (ml) Water (ml) NH₃ (ppm) Negative Control 0 2.01500 FOAMATION ™ 2.0 0 100 Inventive 2.0 0 100

Embodiments

First Embodiment: An animal feed composition comprising a basal animalfeed; and a water-soluble extract of a non-woody plant of the genusHesperaloe comprising at least one saponin.

Second Embodiment: The animal feed composition of the first embodimentwherein the composition is a liquid, a slurry, a dry powder, a drygranular mix, a paste, or a solid.

Third Embodiment: The animal feed composition of the first or the secondembodiment wherein the basal animal feed comprises one or more of cornmeal, corncob grits, soybean meal, alfalfa meal, rice hulls, soybeanmill run, cottonseed oil meal, bone meal, ground corn, corncob meal,wheat middlings, limestone, dicalcium phosphate, sodium chloride, ureaor distillers dried grain.

Fourth Embodiment: The animal feed composition of any one of the firstthrough third embodiments wherein the basal animal feed comprises waterand is a liquid.

Fifth Embodiment: The animal feed composition of any one of the firstthrough fourth embodiments wherein the total amount of saponin rangesfrom about 1 to about 100 g per 100 kg of animal feed.

Sixth Embodiment: The animal feed composition of any one of the firstthrough fifth embodiments wherein the at least one saponin consists ofkammogenin, manogenin, gentrogenin, hecogenin, tigogenin, sarsapogenin,chlorogenin and gitogenin or their corresponding isomer or oxidized orreduced forms and at least one glycosidic moiety selected from glucose,xylose, rhamnose, arabinose, or galactose.

Seventh Embodiment: The animal feed composition of any one of the firstthrough sixth embodiments wherein the at least one saponin is25(27)-dehydrofucreastatin (FIG. 2A), 5(6),25(27)-disdehydroyuccaloisideC (FIG. 2B), 5(6)-disdehydroyuccaloiside C (FIG. 2C), furcreastatin oryuccaloiside C.

Eighth Embodiment: The animal feed composition of any one of the firstthrough seventh embodiments wherein the mass of the water-soluble solidsranges from about 100 to about 1,000 g per kilogram of basal animalfeed.

Ninth Embodiment: The animal feed composition any one of the firstthrough eighth embodiments wherein the water-soluble solids comprisefrom about 10 to about 25 wt % saponin.

Tenth Embodiment: Administering to a non-human animal the animal feedcomposition of any one of embodiments one through nine to reduceenvironmental ammonia and odor, to provide a hypocholesterolemic effect,reduce inflammation, provide an anti-protozoal effect, control parasiticnematodes, promote weight gain or improve feed conversion efficiency.

Eleventh Embodiment: Administered orally to poultry to reduceenvironmental ammonia and odor the animal feed composition of any one ofembodiments one through nine.

Twelfth Embodiment: A urease inhibitor comprising an extract of anon-woody plant of the genus Hesperaloe comprising at least one saponin.

Thirteenth Embodiment: A proteolytic microorganism inhibit comprising anextract of a non-woody plant of the genus Hesperaloe comprising at leastone saponin.

What is claimed is:
 1. An animal feed composition for reducing gaseousemissions, the feed comprising: a. a basal animal feed; and b. anextract from a non-woody plant of the genus Hesperaloe, the extractcomprising 25(27)-dehydrofucreastatin,5(6),25(27)-disdehydroyuccaloiside C, 5(6)-disdehydroyuccaloiside C,furcreastatin, yuccaloiside, or a mixture thereof.
 2. The animal feedcomposition of claim 1 comprising from about 1 to about 100 g of totalsaponins per 100 kg of basal animal feed.
 3. The animal feed compositionof claim 1 wherein extract solids further comprise saccharides,proteins, lipids, or a mixture thereof.
 4. The animal feed compositionof claim 1 wherein the extract substantially free from saccharides,proteins, and lipids.
 5. The animal feed composition of claim 1 whereinthe extract comprises at least about 90% water soluble solids.
 6. Theanimal feed composition of claim 1 wherein the extract comprises lessthan about 2% water insoluble solids.
 7. The animal feed composition ofclaim 1 wherein the extract comprises 25(27)-dehydrofucreastatin,5(6),25(27)-disdehydroyuccaloiside, 5(6)-disdehydroyuccaloiside,furcreastatin and yuccaloiside.
 8. The animal feed composition of claim1 wherein the non-woody plant is selected from Hesperaloe funifera,Hesperaloe nocturne, Hesperaloe parviflora and Hesperaloe chiangii, 9.The animal feed composition of claim 1 comprising from about 100 toabout 1,000 g water soluble solids per kilogram of basal animal feed.10. The animal feed composition of claim 1 wherein the extract comprisefrom about 5 to about 25 wt % saponin.
 11. The animal feed compositionof claim 10 wherein the saponins comprise a mixture of25(27)-dehydrofucreastatin, 5(6),25(27)-disdehydroyuccaloiside C,5(6)-disdehydroyuccaloiside, furcreastatin and yuccaloiside C.
 12. Amethod of reducing gaseous emissions from a non-human animal comprisingadministering thereto a feed composition comprising a basal animal feedand an extract from a non-woody plant of the genus Hesperaloe, theextract comprising at least one saponin.
 13. The method of claim 12wherein the total amount of saponin ranges from about 1 to about 100 gper 100 kg of animal feed.
 14. The method of claim 12 wherein theextract further comprise saccharides, proteins, and lipids.
 15. Themethod of claim 12 wherein the extract is substantially free fromsaccharides, proteins, and lipids.
 16. The method of claim 12 whereinthe at least one saponin is selected from 25(27)-dehydrofucreastatin,5(6),25(27)-disdehydroyuccaloiside C, 5(6)-disdehydroyuccaloiside,furcreastatin and yuccaloiside C.
 17. The method of claim 12 wherein theextract comprise a mixture of 25(27)-dehydrofucreastatin,5(6),25(27)-disdehydroyuccaloiside C, 5(6)-disdehydroyuccaloiside,furcreastatin and yuccaloiside C.
 18. The method of claim 12 wherein thefeed composition comprises from about 100 to about 1,000 g water solublesolids per kilogram of basal animal feed.
 19. The method of claim 12wherein the extract comprises from about 10 to about 25 wt % saponin.20. The method of claim 12 wherein the gas emissions are ammonia orcarbon dioxide.
 21. The method of claim 12 wherein the non-human is abovine, a fowl, a porcine, an ovine or an equine species.
 22. The methodof claim 12 wherein the feed composition is administered orally.
 23. Themethod of claim 12 wherein the feed composition is a liquid, a slurry, adry powder, a dry granular mix, a paste, or a solid.
 24. The method ofclaim 12 wherein the basal animal feed comprises one or more of cornmeal, corncob grits, soybean meal, alfalfa meal, rice hulls, soybeanmill run, cottonseed oil meal, bone meal, ground corn, corncob meal,wheat middlings, limestone, dicalcium phosphate, sodium chloride, ureaor distillers dried grain.
 25. The method of claim 12 wherein the basalanimal feed comprises water and is a liquid.